Morphometric evaluation of the number of exocrine pancreatic cells during early postnatal growth in the rat.

The number of the various cell categories of the exocrine pancreas of the rat was evaluated by morphometric methods in paraffin sections of glands from rats aged 2, 5, 15, 20 and 33 days. The evolution of these cell types could be properly expressed by equations of the type y = aoek.x, where y = cell number, and x = age in days. The time necessary for each cell type to duplicate was thus obtained. The percentages (y') of acinar, intercalated duct and connective tissue cells also proved to be age-dependent and could be expressed by second-degree equations.     

Regulated secretion of mature cathepsin B from rat exocrine pancreatic cells.

By indirect immunofluorescence and immunogold electron microscopy with an antibody that recognizes specifically the two forms of native mature rat cathepsin B (31 kDa and 5:25 kDa) but not the proenzyme, we detected cathepsin B not only in lysosomes of adult rat exocrine pancreatic cells but also in the trans Golgi condensing vacuoles, the zymogen granules and the pancreatic juice in the intralobular ducts. In contrast, immunocytochemistry with an antibody specific for rat cathepsin D showed the latter to be present in the same cells only in lysosomal compartments as expected. The same pattern of labeling with these two antibodies was found in the first zymogen granules to form in 17-day-old fetal rat pancreas. Counts of the extent of immunogold labeling of cathepsin B in the adult exocrine cells showed that the concentration of the enzyme was only two-fold higher in the lysosomal compartments than in the zymogen granules. To confirm these observations, rat pancreatic postnuclear supernatant (PNS), a fraction enriched in zymogen granules and rat pancreatic juice obtained by catheterization of the pancreatic duct, were subjected to 2D gel electrophoresis followed by immunoblotting with the cathepsin B antibody. All three samples contained a 31 kDa protein recognized by the antibody with a pI of about 4.5, the single chain mature form of cathepsin B. We then radiolabeled pancreatic PNS and zymogen granule fractions with benzyloxycarbonyl-Tyr[125I]-Ala-CHN2, an affinity label that covalently binds to the active sites of mature forms of both cathepsin B and cathepsin L. In both PNS and zymogen granule fractions this reagent labeled cathepsin B. Immunoprecipitation experiments showed that the antibody to cathepsin B recognized specifically both the single chain and the double chain mature forms of cathepsin B in the native state. Finally, Northern blots with a cDNA of rat cathepsin B showed that the concentration of cathepsin B mRNA in total pancreatic RNA increased following in vivo stimulation of the exocrine pancreatic cells with optimal doses of cerulein, a cholecystokinin analogue. We conclude that significant amounts of mature cathepsin B are secreted from exocrine pancreatic cells via the apical regulated exocytotic pathway, and we discuss this in terms of models for sorting of proteins to the cores of dense cored secretory granules.     

Localization of protein disulfide isomerase on plasma membranes of rat exocrine pancreatic cells

We investigated immunocytochemically the ultrastructural localization of protein disulfide isomerase (PDI) in rat pancreatic exocrine cells by use of the post-embedding protein A-gold technique. We found that not only the endoplasmic reticulum (ER) and nuclear envelope but also the trans-Golgi cisternae, secretory granules, and plasma membranes were heavily labeled with gold particles. Labeling density of the gold particles in the rough ER and plasma membranes of the exocrine pancreatic cells was twofold and twentyfold greater, respectively, than that of hepatocytes. In the acinar lumen, amorphous material presumably corresponding to the secreted zymogens was also labeled with gold particles. These results suggest that in rat exocrine pancreatic cells a significant amount of PDI is transported to the plasma membrane and secreted to the acinar lumen.     

Comparative studies of the action of long duration alcohol drinking on rat pancreatic exocrine cells

The influence of a long term ingestion of ethanol on the rat exocrine pancreas has been studied by electron microscopy. The comparison of micrographs from alcoholic and non alcoholic pancreas allowed us to compare the different organelles of acinar pancreatic cells. These data have been submitted to factorial analysis of correspondences. The ultrastructural modifications revealed out by this analysis are in good agreement with the hypothesis of an adaptation of exocrine cells to a chronic hyperfunctional state.     

Ultrastructure of rat ovarian interstitial gland cells during pregnancy

Summary The fine structure of the interstitial gland of the rat ovary was studied at estrus and on Days 4, 6, 10, 14 and 18 of pregnancy. At estrus, ovarian interstitial cells have small nuclei with dense irregular clumps of heterochromatin. Mitochondria are small and rod-shaped and have predominantely lamellar cristae. Numerous osmiophilic lipid droplets are present. At Days 4 and 6, nuclear heterochromatin is reduced, and nucleoli are larger and complex. Mitochondria are enlarged and often bizarre-shaped and have tubular cristae. Golgi and smooth endoplasmic reticulum are more conspicuous. At Day 10, prominent ultrastructural features include nuclei with conspicuous heterochromatin, smaller mitochondria with both lamellar and tubular cristae, numerous ribosomes and lipid droplets with decreased osmiophilia. At Days 14 and 18, nuclei have increased heterochromatin, mitochondria are small and have lamellated cristae and an increase in the size and number of lipid droplets occurs. These observations suggest that steroidogenic activity of interstitial cells is highest during the first half of pregnancy and regresses during the last half. It is suggested that the interstitial gland is an important ovarian source of pregnancy hormone(s) during the first half of gestation and that LH may modulate steroidogenic activity in this ovarian component.     

Ultrastructure of cells of rat lymphatic organs during continuous irradiation

Changes in the ultrastructures of lymphatic organs of rats observed from the 2nd to 22nd day following continuous irradiation with gamma rays at a daily dose of 115 mGy (exposition: 12 R/day) are described. The maximum of destructive changes in lymphocytes was observed on the 11th to the 14th day of irradiation. A gradual balance between dystrophic and regenerative processes was achieved on the 18th day. In this connection to correlation could be determined between the ultrastructural changes described and the fluctuations of lymphocyte numbers in the lymphatic organs and peripheral blood during continuous irradiation.     

Caseinomacropeptide specifically stimulates exocrine pancreatic secretion in the anesthetized rat

The effect of caseinomacropeptide (CMP) (the [106-169] fragment of kappa-casein produced during digestion of milk protein), was studied in anesthetized rats using bile diversion for a pure pancreatic juice collection system. Intraduodenal administration of CMP induced a dose-related specific stimulation of pancreatic secretion which was nearly abolished by devazepide, atropine, hexamethonium, vagotomy or perivagal capsaicin pretreatment. Moreover, CMP did not inhibit in vitro trypsin activity. These results demonstrate that CMP is more likely to stimulate pancreatic secretion specifically through cholecystokinin release and activation of a vago-vagal cholinergic reflex loop than by inhibition of luminal trypsin, in anesthetized rats.     

The effect of insulin on amino acid incorporation into exocrine pancreatic cells of the rat.

The rate of incorporation of radioactive leucine per cell in the acinar pancreatic cells of the rat increases by 50 per cent within one hour after subcutaneous administration of insulin, an effect that lasts for at least one more hour. The rate of incorporation has been measured by quantitative radioautography and by determination of the radioactivity per mug DNA in TCA-precipitable material from tissue homogenates. The capacity for amino acid (leucine and lysine) incorporation as measured by incubating pancreatic fragments in vitro is not enhanced by insulin treatment of the rat in vivo during one or more hours. Insulin was found to lower the serum concentration of most amino acids significantly, leucine by 50 per cent. The apparent effect of insulin on the incorporation of radioactive leucine in vivo can be explained by the difference in the specific radioactivity of the circulating amino acid in the treated rats as compared to the untreated ones. A change in amino acid concentration in the serum may likewise be the explanation of the decrease in amino acid incorporation rate in alloxan diabetic rats. The absence of a short term effect of insulin on the rate of protein synthesis does not exclude a long term effect as suggested by the higher rate of incorporation in the cells of peri-insular acini.     

Membrane detachment and release of the major membrane glycoprotein of secretory granules in rat pancreatic exocrine cells

The major glycoprotein of pancreatic zymogen granule membranes (GP-2) was detected in the medium of acinar cell suspensions from rat pancreas. Its release from the cells was studied in pulse-chase metabolic labeling experiments with radioactive methionine. GP-2 (apparent Mr = 80 000) was found to be processed to a form of slightly lower apparent Mr (75 000) after about 4 h chase. At about the same time this smaller form of GP-2 appeared in the medium. These results are in accordance with earlier findings in vivo. At different chase times acinar cells were extracted with Triton X-114 to separate water-soluble proteins from membrane-associated (hydrophobic) proteins. This experiment showed that GP-2 is slowly converted from a membrane-bound glycoprotein to a soluble glycoprotein after its reduction in apparent molecular mass, causing its detachment from the membrane. Further analysis indicated that the detachment process may occur at the zymogen granule membrane as well as the plasma membrane. Immunocytochemistry on ultrathin cryosections of pancreatic tissue showed that GP-2 is localized on zymogen granule membranes, plasma membranes and in the acinar lumen. Although in much smaller quantities, GP-2 is also present in the granule content. Thus, in summary, GP-2 is synthesized as a true membrane glycoprotein which is gradually processed to a soluble species and is found in the secretion.     

Immunocytochemical localization of exocrine enzymes in rat pancreatic acinar carcinoma

Ten pancreatic secretory proteins have been demonstrated in differentiated pancreatic acinar carcinoma cells by the protein A-gold immunocytochemical approach. The high resolution of the technique has allowed for the localization of the different proteins in the cellular compartments involved in protein secretion: RER, Golgi and secretory granules. The quantitative evaluation of the labeling for amylase has demonstrated the presence of an increasing gradient in the intensity from the RER to the Golgi and to the secretory granules which may reflect the process of protein concentration along the secretory pathway. These results, together with those obtained using the pulse-labeling autoradiographic approach, demonstrate that differentiated acinar carcinoma cells are capable of processing secretory proteins. When intensities of labeling obtained for different proteins on acinar carcinoma cells were compared to those obtained on normal pancreatic acinar cells, major differences were observed for some proteins. In addition, studies performed on the pancreatic tissue of the tumor-bearing animals have shown the presence of morphological alterations in the acinar cells.     

Effect of colonic distension on exocrine pancreatic secretion of the conscious rat

In the conscious rat the increasing distension of the colon (from 2 to 6 ml) induced a slight decrease of exocrine pancreatic secretion which was more important for protein output than for volume. Nevertheless this inhibitory effect was never significant even with the important distension of 6 ml which induced a visible uneasiness in the animal.     

Morphological changes in the rat exocrine pancreas after pancreatic duct ligation

In the present study, morphological changes of the exocrine pancreas in rats after pancreatic duct ligation were examined with light microscopy (hematoxylin-eosin, TUNEL, and PCNA staining) and scanning electron microscopy in order to elucidate the effects of increased pancreatic duct pressure. On the fifth day after pancreatic duct ligation, ductular proliferation, periductal fibrosis, and disappearance of acini were observed. TUNEL and PCNA staining demonstrated many apoptotic acinar cells and proliferating ductal cells immediately after ligation, which reached a maximal number on the 2nd or 3rd day. Tortuous or helical interlobular pancreatic ducts with inner surfaces containing many crater-like depressions and long cilia were found after ligation. These changes were almost identical to those observed in the pancreatic tissue of model chronic pancreatitis rats, WBN/Kob rats, and stroke-prone spontaneously hypertensive (SHRSP) rats. In summary, the morphological changes observed after pancreatic duct ligation were similar to those of chronic pancreatitis, therefore, the characteristic changes of pancreatic ducts observed in chronic pancreatitis may be caused by increased pancreatic duct pressure.     

Condensation-sorting events in the rough endoplasmic reticulum of exocrine pancreatic cells

In guinea pig exocrine pancreatic cells intracisternal granules (ICGs) occur at a low frequency within the lumen of the RER. By starving and refeeding guinea pigs or injecting them in CoCl2 solution, the number of these granules is greatly increased. We show here that ICGs contain the complete set of secreted pancreatic digestive enzymes and proenzymes. Two other soluble proteins in the lumen of the RER, GRP 78/BiP and protein disulphide isomerase (PDI), are specifically excluded from ICGs. The formation of ICGs, which occurs without acidification of the RER cisternae, is therefore a sorting event involving the cocondensation of a complete set of secretory enzymes and proenzymes, which for brevity we refer to collectively as the zymogens. With the exception of approximately 50% of the RNase, the zymogens in ICGs are covalently cross-linked by intermolecular disulphide bonds. The synthesis of all three resident ER cisternal proteins--PDI, GRP 78/BiP, and GRP 94--with the carboxy-terminal sequence KDEL, is induced in response to the accumulation of massive amounts of misfolded secretory protein in the ICGs in the lumen of the RER. After injection of rats with large doses of parachlorophenylalanine-methylester, crystals form in the lumen of the RER. We show that these crystals appear to be a lattice of amylase with the other zymogens incorporated between the layers. Both GRP 78/BiP and PDI are excluded from these crystals. The formation of these amylase crystals within the RER and the inclusion of other zymogens is, therefore, also a sorting event. These data establish that in exocrine pancreatic cells zymogens can cocondense in the RER into either amorphous aggregates or crystals that exclude other soluble RER proteins. This demonstrates that cocondensation is a mechanism capable of sorting zymogens within the secretory pathway.     

Ultrastructure of cells of the rat entero-endocrine system during exposure to cholera toxin

Horse radish peroxidase-labelled cholera toxin (choleragen) binds to the receptors of entero-endocrine cell (Ec-cells) plasmalemma. Unlike columnar epitheliocytes, the effect of choleragen on Ec-cells.     

Inhibitory effect of pancreastatin on pancreatic exocrine secretion in the conscious rat

The effect of newly discovered pancreastatin on pancreatic secretion stimulated by a diversion of bile-pancreatic juice (BPJ) from the intestine was examined in the conscious rat. Exogenous pancreastatin infusion (20, 100 and 200 pmol/kg.h) inhibited pancreatic protein and fluid outputs during BPJ diversion in a dose-dependent manner. Pancreastatin did not affect plasma cholecystokinin (CCK) concentrations. Pancreastatin (100 pmol/kg.h) inhibited CCK-stimulated pancreatic secretion, but did not inhibit secretin-stimulated pancreatic secretion. Pancreastatin alone, however, did not affect basal pancreatic secretion. In contrast, pancreastatin (10(-10)-10(-7)M) did not suppress CCK-stimulated amylase release from isolated rat pancreatic acini. These results indicate that pancreastatin has an inhibitory action on exocrine function of the pancreas. This action may not be mediated by direct mechanisms and nor via an inhibition of CCK release. It is suggested that pancreastatin may play a role in the regulation of the intestinal phase of exocrine pancreatic secretion.     

Volume changes during mitosis in pancreatic acinar cells of the rat

The volume of interphasic and mitotic pancreatic acinar cells of 20-day-old rats was estimated by analysis of drawings from Araldite-embedded serial thick sections. Assuming the average interphase cell volume to be 100%, the volume of prophase cells was 145%, reaching a volume of 180% during anaphase. Postmitotic acinar cell volume was 74%.